ABSTRACT Drug addiction, including cocaine-abuse disorder, represents a major public health issue, characterized by compulsive drug seeking and increased propensity to relapse after abstinence. Relapse to cocaine seeking is triggered by environmental cues strongly associated with the rewarding effects of the drug itself. The neuronal encoding of cocaine-associated cues at a single-cell level remains unclear, limiting our understanding of how cocaine-paired cues trigger drug seeking behavior. Via its constituent dopamine D1- and D2- receptor expressing medium spiny projection neurons (MSNs), the nucleus accumbens core (NAcore) plays a key role in encoding cue-reward associations and triggering relapse to cocaine seeking behavior. The NAcore integrates multiple cortical and allocortical inputs. In particular, the activation of afferent glutamatergic projections from the prelimbic cortex (PL) are necessary for cue-induced reinstatement of cocaine seeking. Using chemogenetic and optogenetic manipulations on D1- and D2-MSNs, previous studies have shown opposite roles of these neuronal subtypes on cue-induced reinstatement of cocaine seeking in rodent models of self-administration. Therefore, I hypothesize that cocaine seeking during context/cue-reinstatement and refraining from seeking during extinction training are associated with temporally and spatially distinct neuronal ensembles of D1- and D2-MSNs that are differentially modulated by PL-NAcore projections. I will address this hypothesis by quantifying Ca2+ activity in D1-MSNs (Aim 1) and D2-MSNs (Aim 2) during cocaine-seeking and extinction, with and without inhibiting PL- efferents to NAcore. To investigate the functional role of D1- and D2-MSNs during cocaine seeking, we will record single-cell Ca2+ dynamics from D1- and D2-MSNs using a head-mounted miniature microscope and virally expressed Cre-dependent Ca2+ indicator (GCaMP6f) in D1- and D2-cre transgenic mice while undergoing cocaine self-administration, extinction training and cue-induced reinstatement, We will further inhibit PL-NAcore projections during drug seeking tests (post abstinence and during cue-induced reinstatement) using virally expressed inhibitory Gi-DREADDs. Recorded Ca2+ dynamics will be analyzed using advanced statistical models and clustered to isolate the neuronal ensembles associated with context/cue-induced drug seeking and refraining from seeking during extinction. The information obtained from this research will isolate neuronal ensembles that encode cocaine-seeking, as well as the mechanism(s) by which PL-NAcore projections affect these ensembles to regulate cue-induced reinstatement. This fellowship will train me in experimental design, scientific writing, and cutting-edge techniques to understand the brain circuits underlying drug addiction, including viral transfection and micro-endoscopic analysis of Ca2+ activity in transgenic mice trained to self-administer and reinstate to drug- associated cues and contexts.